Remarkable positional (regio)specificity of xanthine oxidase and some dehydrogenases in the reactions with substituted benzaldehydes.

Upon an increase in the size of the substituent, the reactivity of xanthine oxidase to ortho-substituted benzaldehydes drastically decreases while that to para-substituted benzaldehydes does not change significantly. The enzyme exhibits this regiospecificity with respect to both electron-withdrawing substituents (e.g., halogens) and electron-donating ones (alkyls and alkoxyls). Xanthine oxidase-catalyzed oxidation of m- and p-nitrobenzaldehyde is more than 300-times faster than that of the o-isomer, whereas the rates of their non-enzymatic oxidation are comparable, as are the rates of the enzymatic oxidation of p- and o-nitrocinnamaldehyde. These and other findings of this work indicate that the discovered positional specificity of xanthine oxidase is due to steric hindrances in the reaction of the enzyme's active center with the aldehyde moiety having a bulky substituent in its close proximity. Such regiospecificity of the enzyme exists regardless of the nature of the electron acceptor used and can be employed for the separation of mixtures of positional isomers of substituted benzaldehydes. A marked positional specificity in the xanthine oxidase-catalyzed oxidation of substituted benzaldehydes appears to be a rather general phenomenon: three other enzymes tested, alcohol dehydrogenases from horse liver and yeast and aldehyde dehydrogenase from yeast, all follow a similar pattern in the reactions with para- and ortho-substituted halobenzaldehydes.